Antibacterial Vaccine Strategies
Exported proteins in bacteria participate in many diverse and essential cell functions such as motility, signal transduction, macromolecular transport and assembly, and the acquisition of essential nutrients. For pathogenic bacteria, many exported proteins are virulence determinants that function as adhesions to colonize and thus infect the host or as toxins to protect the bacteria against the host's immune system [International Patent Publication No. WO 95/06732, published Mar. 9, 1995 by Masure et al., which is specifically incorporated herein by reference in its entirety, for a review, see Hoepelman and Tuomanen, Infect. Immun., 60:1729-33 (1992)]. However, other exported proteins may not directly mediate adhesion.
Since the development of the smallpox vaccine by Jenner in the 18th century, vaccination has been an important armament in the arsenal against infectious microorganisms. Prior to the introduction of antibiotics, vaccination was the major hope for protecting populations against viral or bacterial infection. With the advent of antibiotics in the early 20th century, vaccination against bacterial infections became much less important. However, the recent insurgence of antibiotic-resistant strains of infectious bacteria has resulted in the reestablishment of the importance of anti-bacterial vaccines.
One possibility for an anti-bacterial vaccine is the use of killed or attenuated bacteria. However, there are several disadvantages of whole bacterial vaccines, including the possibility of a reversion of killed or attenuated bacteria to virulence due to incomplete killing or attenuation and the inclusion of toxic components as contaminants.
Another vaccine alternative is to immunize with the bacterial carbohydrate capsule. Presently, vaccines against Streptococcus pneumoniae employ conjugates composed of the capsules of the 23 most common serotypes of this bacterium. these vaccines are ineffective in individuals most susceptible to pathological infection--the young, the old, and the immune compromised--because of its inability to elicit a T cell immune response. A recent study has shown that this vaccine is only 50% protective for these individuals [Shapiro et al., N. Engl. J. Med. 325:1453-60 (1991)].
An alternative to whole bacterial vaccines are acellular vaccines or subunit vaccines in which the antigen includes a bacterial surface protein. These vaccines could potentially overcome the deficiencies of whole bacterial or capsule-based vaccines. Moreover, given the importance of exported proteins to bacterial virulence, these proteins are an important target for therapeutic intervention. Of particular importance are proteins that represent a common antigen of all strains of a particular species of bacteria for use in a vaccine that would protect against all strains of the bacteria. However, to date only a small number of exported proteins of Gram positive bacteria have been identified, and none of these represent a common antigen for a particular species of bacteria.
Recently, apparent fusion proteins containing PhoA were exported in species of Gram positive and Gram negative bacteria (Pearce and Masure, 1992, Abstr. Gen. Meet. Am. Soc. Microbiol. 92:127, abstract D-188). This abstract reports insertion of pneumococcal DNA upstream from the E. coli phoA gene lacking its signal sequence and promoter in a shuttle vector capable of expression in both E. coli and S. pneumoniae, and suggests that similar pathways for the translocation of exported proteins across the plasma membranes must be found for both species of bacteria.
In previous studies, use of random translational gene fusions (PhoA mutagenesis) to identify and alter exported proteins in Streptococcus pneumoniae provided insight into putative exported proteins [Pearce et al., Mol. Microbiol., 9:1037 (1993); International Patent Publication No. WO 95/06732, published Mar. 9, 1995; U.S. patent application Ser. No. 08/116,541, filed Sep. 1, 1993; U.S. patent application Ser. No. 08/245,511, filed May 18, 1994]. Coupling this gene fusion technology to bioactivity assays for pneumococcal adherence, the primary goal was to genetically identify and characterize immunogenic adhesion virulence determinants to eucaryotic cells that define the bacteria-host relationship and thus serve as vaccine candidates. Over 25 loci that effect adherence have been identified as determinants of virulence.
In addition, the molecular mechanism of pathogenesis caused by pneumococcus are beginning to be defined [Cundell, et al., Infect. Immun. 63:2493-2498 (1995); Wizemann, et al., Proc. Natl. Acad. Sci. USA (1996); Cundell, et al., J. Cell Biol. S18A:45 (1994); Spellerberg, et al., Mol. Microb. (1996)]. The results of these efforts shows that many bacterial components participate in a complex coordinated process to cause disease. However, it is also apparent that this strategy has produced only a few potential vaccine candidates.
Of note in the search for exported pneumococcal proteins that might be attractive targets for a vaccine is pneumococcal surface protein A (PspA) [see Yother et al., J. Bacteriol., 174:610 (1992)]. PspA has been reported to be a candidate for a S. pneumoniae vaccine as it has been found in all pneumococci to date; the purified protein can be used to elicit protective immunity in mice; and antibodies against the protein confer passive immunity in mice [Talkington et al., Microb. Pathog. 13:343-355 (1992)]. However, PspA demonstrates antigenic variability between strains in the N-terminal half of the protein, which contains the immunogenic and protection eliciting epitopes (Yother et al., supra). This protein does not represent a common antigen for all strains of S. pneumoniae, and therefore is not an optimal vaccine candidate.